KR100250090B1 - Cdna encoding a human c6 beta-chemokine, leukotactin-2, and a process for preparing a recombinant leukotactin-2 - Google Patents

Abstract

The present invention utilizes a cDNA encoding a novel protein (leukotactin-2: Lkn-2) belonging to C6 β-chemokine, isolated from a human mononuclear cell line (monocytic cell line), an expression vector comprising the same, an electric expression vector The present invention relates to a method for producing an electrical protein that attracts subsets of peripheral blood leukocytes. The open reading frame of Lkn-2 cDNA of the present invention encodes a total of 137 amino acids, including a signal peptide consisting of 21 amino acids, of which the molecular weight of the mature protein consisting of 116 amino acids is estimated to be 12,500 Daltons. Recombinant Lkn-2, purified by expressing the aforementioned Lkn-2 cDNA in recombinant E. coli or insect cells, significantly inhibits colony formation, has chemotaxis that attracts lymphocytes, monocytes and neutrophils, and hMIP-1α and RANTES It has been shown to bind to the receptor of binding CCR1. The recombinant Lkn-2 protein can be used for antibody production, treatment during HIV-1 infection, bone marrow hepatocyte protection during chemotherapy or radiation therapy, leukemia inhibitor, and the like.

Description

Method for preparing cDNA and recombinant Lkn-2 of C6 β-chemokine Lkn-2 (leukotactin-2) isolated from humans

The present invention relates to a method for producing cDNA and electric protein of novel C6 β-chemokine isolated from humans. More specifically, the present invention provides a peripheral blood leukocyte using a cDNA encoding a novel protein belonging to C6 β-chemokine isolated from a human mononuclear cell line, an expression vector comprising the same, and an electric expression vector. The present invention relates to a method for producing an electrical protein that attracts subsets of peripheral blood leukocytes.

Chemokines belong to a family of cytokines consisting of low molecular weight baseline proteins, which have four cysteines in common, depending on whether the first two cysteines are adjacent to each other or amino acids are intermediate. α), CC (β), γ (C) and CX ₃ C, which are classified into four subfamily (Baggiolini, M. and Dahinden, CA, Immunol. Today, 15: 127 (1994); Kelner, SG et al., Science, 266: 1395 (1994); Bazan, JF et al., Nature, 385: 640 (1997)). The genes of the chemokine subfamily are clustered on the same chromosome, for example, α-chemokine genes on the human 4q12-21 chromosome and β-chemokine genes on the human 17q11-32 chromosome and mouse 11 chromosome. Located.

These chemokines have been reported to exhibit biological activities such as HIV-inhibiting, immunomodulatory, leukocyte migration, and inhibiting division of hematopoietic stem cells (Cocchi, F. et al., Science, 270: 1811). (1995); Wolpe, SD et al., J. Exp. Med., 167: 570 (1988); Graham, GJ et al., Nature, 344: 442 (1990); Broxmeyer, HE et al., Blood, 76: 1110 (1990); Youn, B.-S. et al., J. Immunol., 155: 2661-2667 (1995).

In addition, chemokines bind to transmembrane domain G protein-coupled receptors to activate leukocytes, some of which are reported to be used as coreceptors in HIV-1 infection. Oh, K.-O. et al., J. Immunol., 147: 2978 (1991); Alkbatih, G. et al., Science, 272: 1955 (1996). For example, there are eight receptor subtypes of β-chemokine (CC chemokine), namely CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7 and CCR8, among which CCR1, CCR2, CCR3 and CCR5 are found in various substances. CCR4, CCR6, CCR7 and CCR8 have been shown to exhibit high affinity for one substance, although they exhibit binding ability.

Nine chemokines belonging to the β-chemokine subfamily have been identified (Wild, SD et al., J. Exp. Med., 171: 1301 (1990); Modi, WS et al., Hum. Genet., 84 : 185 (1990)), among them murine MRP-1 (macrophage inflammatory protein) -related protein-1 (MIP) (Orlofsky, A. et al., Cell Regul., 2: 403 (1991)). ) And murine MRP-2 (Youn, B.-S. et al., J. Immunol., 155: 2661 (1995)) have two more cysteine residues, Disulfide bonds are possible and the N-terminal sites are quite long, distinguishing them from the remaining seven chemokines. Thus, these MRPs were classified as C6 β-chemokines.

Currently only mMRP-1 and mMRP-2 have been reported, but due to the potential efficacy of MRPs that can be applied for the treatment of HIV-1 infection or for the protection of bone marrow hepatocytes during chemotherapy or radiation therapy, Separation and characterization of MRP is urgently needed.

Thus, the inventors of the present invention sought to isolate a novel MRP gene from various human cell lines. As a result, we isolated MRP cDNA belonging to C6 β-chemokine from human mononuclear cell lines, and determined the base sequence and amino acid sequence inferred therefrom. Confirming that they are novel, and furthermore, electric MRP cDNA was expressed in recombinant E. coli or insect cells, and confirmed that the expressed recombinant MRP has a chemotaxis that attracts sebsets (lymphocytes, monocytes, neutrophils) of peripheral blood leukocytes, The present invention has been completed. Thus, hereinafter, the electric MRP cDNA isolated from human was named "Lkn-2 (leukotactin-1) cDNA", and recombinant MRP was named "recombinant Lkn-2".

After all, the first object of the present invention is to provide cDNA of the novel C6 β-chemokine (Lkn-2) and amino acid sequences inferred therefrom.

It is a second object of the present invention to provide an expression vector comprising the Lkn-2 cDNA and a microorganism transformed therewith.

It is a third object of the present invention to provide a method for preparing recombinant Lkn-2 from an electrotransformed microorganism.

Figure 1 (A) shows the amino acid sequence which determines the nucleotide sequence of the 202 bp DNA fragment and infers therefrom.

Figure 1 (B) is a comparison of the amino acid sequence of 87 to 110 of Lkn-1 exon and mMRP-2.

2 is a photograph showing the result of reverse transcriptase-polymerase chain reaction (RT-PCR) of total cell RNA isolated from various human cell lines using Lkn-1 primer and actin primer.

3 shows the cDNA nucleotide sequence of Lkn-2 and the amino acid sequence inferred therefrom.

4 (A) is a graph showing the extent to which recombinant Lkn-2 inhibits colonies formed by CFU-GM.

4 (B) is a graph showing the extent to which recombinant Lkn-2 inhibits colonies formed by BFU-E.

4 (C) is a graph showing the extent to which recombinant Lkn-2 inhibits colonies formed by CFU-GEMM.

5 (A) is a graph showing the chemotaxis of recombinant Lkn-2 and RANTES on lymphocytes.

5 (B) is a graph showing the chemotaxis of recombinant Lkn-2 and hMIP-1α against monocytes.

5C is a graph showing the chemotaxis of recombinant Lkn-2 and IL-8 against neutrophils.

FIG. 6 (A) shows the relative fluorescence measured by RANTES applied to lymphocytes against a series of stimuli of recombinant Lkn-2.

Figure 6 (B) shows the relative fluorescence measured for a series of stimuli of recombinant Lkn-2 and RANTES applied to lymphocytes.

Figure 6 (C) shows the relative fluorescence measured for a series of stimuli of hMIP-1α and recombinant Lkn-2 applied to monocytes.

Figure 6 (D) shows the relative fluorescence measured for a series of stimuli of recombinant Lkn-2 and hMIP-1α applied to monocytes.

Figure 6 (E) shows the relative fluorescence measured for a series of stimuli of IL-8 and recombinant Lkn-2 applied to neutrophils.

Figure 6 (F) shows the relative fluorescence measured for a series of stimuli of recombinant Lkn-2 and IL-8 applied to neutrophils.

7 (A) shows the relative fluorescence measured for a series of stimuli of IL-8, hMIP-1α and hMIP-1α applied to HOS cells expressing CCR1 receptor.

7 (B) shows the relative fluorescence measured for a series of stimuli of hMIP-1α and recombinant Lkn-2 applied to HOS cells expressing CCR1 receptor.

Figure 7 (C) shows the relative fluorescence measured for a series of stimuli of recombinant Lkn-2 and hMIP-1α applied to HOS cells expressing CCR1 receptor.

8 is a graph showing the result of the reaction according to the concentration of Lkn-2 and hMIP-1α.

Hereinafter, the present invention will be described in more detail.

In order to isolate the novel Lkn-2 gene from human cell lines, we first cloned the exon sites of Lkn-1 gDNA to be used for the provision of probes. That is, the entire human genome genomic DNA was digested with HindIII restriction enzymes, fractionated on agarose gels, and labeled with mMRP-2 cDNA labeled with ^32- P (Youn, BS et al., J. Immunol., Southern blot analysis using 155: 2661 (1995)) as a probe yielded a 7.0 kb DNA fragment hybridized with mMRP-2. Then, to separate the exon sequence of Lkn-1 gDNA from the 7.0 kb HindIII DNA fragment, the human genomic DNA was completely cleaved with HindIII and fractionated on agarose gel to separate the 7.0 kb DNA fragment and then the fragment. Was inserted into the vector, introduced into the host cell, and cultured to select colonies exhibiting hybridization with the mMRP-2 cDNA probe. Then, a 7.0 kb DNA fragment was isolated from the colony, digested with Alu I, and cloned a 202 bp DNA fragment that hybridized with mMRP-2 cDNA. The base sequence of this fragment was determined to identify the exon moiety translated into introns and amino acids (see Korean Patent Application No. 96-43477).

Then, to clone the novel Lkn-2 cDNA from human cell lines, we devised a Lkn-1 polymerase chain reaction (PCR) primer capable of amplifying 100 bp from the Lkn-1 exon sequence identified above, RT-PCR (reverse transcriptase-polymerase chain reaction) was used as a template for the total cell RNA isolated from the cell line, and cell lines for detecting mRNA of Lkn-1 were selected. One of these selected cell lines, a cDNA library of human mononuclear THP-1 cell line stimulated with interleukin-4 (IL-4), was prepared, while total cell RNA of the electric THP-1 cell line was used as a template. Then, 100-bp DNA fragment of Lkn-1 exon was prepared by RT-PCR using the Lkn-1 PCR primer. As a result of hybridizing the cDNA library of the THP-1 cell line prepared above and Lkn-1 exon using a 100 bp DNA fragment as a probe, Lkn-2 cDNA showing a positive reaction was found.

As a result of determining the nucleotide sequence of the Lkn-2 cDNA and the amino acid sequence inferred therefrom, Lkn-2 cDNA is a novel one that has not been reported so far, and the Lkn-2 cDNA open reading frame is 21 amino acids. A total of 137 amino acids were encoded, including the signal peptide. Among them, the molecular weight of the mature protein bill consisting of 116 amino acids was estimated to be 12,500 Daltons. In addition, Lkn-2 cDNA does not have an N-glycosylation site and, in addition to the four cysteine residues common to the β-chemokine family, mMRP-2, mMRP-2 and Lkn-1 (see: Belonging to the C6 β-chemokine family which additionally has two cysteine residues found in the same applicant filed "CDNA of human isolated C6 β-chemokine Lkn-1 (leukotactin-1) and recombinant microorganisms expressing it"). Could.

The cloned novel Lkn-2 cDNA was inserted into an expression vector, introduced into a host cell such as Escherichia coli or insect cells, and then expressed, followed by pure separation of the expressed Lkn-2.

Purified recombinant Lkn-2 significantly inhibits colony formation by granulocyte-macrophages and the like in a concentration-dependent manner, exhibits strong chemotaxis against human peripheral blood neutrophils, monocytes and lymphocytes, and induces calcium efflux from these cells do. In particular, recombinant Lkn-2 shares CCR1 receptors with human macrophage inflammatory protein (hMIP) -1α, a regulated activated normal Tcell expressed sequence (RANTES), and is an agonist of CCR1 receptors that is stronger than the known hMIP-1α. Turned out to be.

The recombinant Lkn-2 of the present invention having the above-mentioned characteristics can be used for antibody production, treatment during HIV-1 infection, bone marrow hepatocyte protection during chemotherapy or radiation therapy, leukemia inhibitor, and the like.

Meanwhile, in the amino acid sequence of the present invention, "functionally equivalent" means Gly, Ala in the amino acid sequence of Lkn-2 protein; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; And all proteins substituted with a combination such as Phe, Tyr. In addition, in the base sequence of the present invention, the term "functional equivalent" refers to all genes having a base sequence capable of providing the amino acid of the above combination in the base sequence of the Lkn-2 cDNA gene.

Hereinafter, the present invention will be described in detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1

Exon Cloning of Lkn-1 Genomic DNA

To clone a genomic gene with sequencing homology with mMRP-2 in humans, the entire human genomic DNA was first cleaved with BamH I, EcoR I, HindIII, Pst I, Xba I and Xho I, and then 0.8%. Fractionation on agarose gel and Southern blot analysis using a ^32- P labeled mMRP-2 cDNA as a probe yielded a 7.0 kb HindIII fragment which showed positive reaction.

To prepare a sublibrary of the HindIII fragment, 100 μg of human genomic DNA was first cut completely with HindIII, fractionated at 20V on 1% agarose gel for 16 hours, and then separated and purified around DNA of 7.0 kb. , And was inserted into dephosphorylated pBluescript SK ⁺ vector (Stratagene, USA) digested with HindIII. The recombinant vector thus prepared was introduced into a GIBCO-BRL, USA transformable cell by the electroporation method, cultured in a solid medium, and then grown to about 2 × 10 ⁵ colonies by mMRP. Hybridization with the -2 probe revealed that only one colony showed hybridization positive reactions, which contained 7.0 kb of insert DNA.

To separate the exon sequence from the 7.0 kb genomic DNA fragment, pBluescript SK ⁺ vector containing 7.0 kb of insert DNA was isolated from the colonies that showed hybridization positive reaction, cut with Alu I and digested with 1.5% agarose gel. Fractionation on phase was followed by Southern blot analysis to clone a 202 bp DNA fragment that hybridized with mMRP-2 cDNA.

The base sequence of the 202 bp DNA fragment was determined to identify an exon moiety that is translated into introns and amino acids (see Figure 1 (A)). This exon region shows 50% homology with the amino acid sequences of mMRP-2 87-110 and is also the second of two additional cysteines ("*") that are common to both mMRP-1 and mMRP-2. It was confirmed that the mark) is preserved in Lkn-1 (see also Figure 1 (B)). In the first (B), square parts represent amino acids conserved between Lkn-1 and mMRP-2, respectively.

Example 2

Cloning of Lkn-2 cDNA

First, RT-PCR against human mononuclear cell THP-1 cell line (ATCC TIB202), macrophage cell line U937 (ATCC CRL1593) and HL60 cell line (ATCC CCL240) to select human cell lines expressing Lkn-1 mRNA. Was performed. That is, the first chain cDNA is synthesized from poly (A) ⁺ RNA by Superscript (Life Technologies, USA), and the product cDNA-RNA fusion chain (cDNA-RNA hybrid) is separated from each other, and double-chain cDNA by PCR. After being amplified with, one strand of chain was amplified by a primer designed from the Lkn-1 exon sequence and the other strand was amplified by an actin primer. Each PCR product was then mixed in equal amounts and electrophoresed on a 2.0% agarose gel (see Figure 2). At this time, the Lkn-1 PCR primers used the following oligonucleotides capable of amplifying 100 bp DNA fragments designed from the Lkn-1 exon sequence disclosed in FIG. 1 (A):

Forward primer 5'-CCGGAAATTCCCAGATTCCTCACCAAGAAG-3 ';

Reverse primer 5'-CGCGGATCCCTTTTTCATGCAATCCTGAACTCCCGG-3 '.

In addition, actin mRNA was used as a control of the RT-PCR, and the actin PCR primers used the following oligonucleotides capable of amplifying 500 bp DNA fragments.

Forward primer 5'-CACGTCACACTTCATGATGG-3 ';

Reverse primer 5'-ATGTTTGAGACCTTCAACAC-3 '.

In FIG. 2, the control group represents the RNA of the unstimulated cell line, and the RMA-LPS represents the RNA of the cell line stimulated for 48 hours with 20ng / ml of PB (phorbal myristic acetate) and 5μg / ml of lipopolysaccharide (LPS). , IL-4 represents RNA of cell lines stimulated for 48 hours with 100 μg / ml IL-4, PMA + LPS + IL-4 represents 20ng / ml PMA, 5μg / ml LPS and 100μg / ml IL- 4 represents the RNA of the cell line stimulated for 48 hours, and lane M represents the 100 bp ladder as the DNA size marker, respectively. As shown in FIG. 2, Lkn-1 mRNA was induced in THP-1 cell line and U937 cell line stimulated with IL-4 or PMA + LPS, and was always expressed in HL60 cell line.

Based on the experimental results described above, we tried to clone Lkn-2 cDNA from human monocytes THP-1 cell line stimulated with IL-4. First, human mRNA was isolated from THP-1 cell line stimulated for 48 hours with 100 μg / ml IL-4, and reverse transcribed into double-chain cDNA using Time Saver cDNA kit (Pharmacia Biotech, USA). By attaching a BstX I adapter (Invitrogen, USA) to it, fractionated on agarose gel, and separating 0.5 c or more cDNA and inserting it into a PRc / CMV (Invitrogen, USA) vector cleaved with BstX I, a THP-1 cell line CDNA library was prepared.

In addition, a probe to be used for cloning Lkn-2 cDNA was prepared as follows: total cell RNA of THP-1 cell line stimulated with 100 μg / ml IL-4 for 48 hours as a template, and the Lkn-1 PCR. RT-PCR was performed using the primers.

Using a 100 bp DNA fragment of the amplified Lkn-1 exon as a probe, hybridization reaction with the cDNA library of the THP-1 cell line prepared above, a novel cDNA having a sequence different from that of Lkn-1 cDNA (see below) Example 3), ie Lkn-2 cDNA, was found and isolated.

Example 3

Sequence analysis of Lkn-2 cDNA

The base sequence of the cloned Lkn-2 cDNA in Example 2 and the amino acid sequence inferred therefrom are shown in FIG. In Figure 3, the underlined portion is a signal peptide; The square portion contains six conserved cysteine residues; And (***) indicates a translation termination site.

As shown in FIG. 3, the open reading frame of Lkn-2 cDNA consists of a total of 137 amino acids consisting of a signal peptide of 21 amino acids and a mature protein of 116 amino acids estimated to have a molecular weight of 12,500 Daltons. In addition, the inferred Lkn-2 amino acid sequence shows 73% homology with Lkn-1, does not have an N-glycosylation site, and in addition to the four cysteine residues common to the β-chemokine family, mMRP-1, Further two cysteine residues found in mMRP-2 and Lkn-1 (see, eg, the cDNA of C6 β-chemokine Lkn-1 isolated from humans (leukotactin-1) and recombinant microorganisms expressing it)) The branches belonged to the C6 β-chemokine family.

In addition, the inferred Lkn-2 amino acid sequence differs from Lkn-1 in that it additionally has 18 amino acids immediately preceding the C-C motif (71 th and 72 th amino acids). Therefore, Lkn-2 has an unusually long N-terminus compared to other β-chemokines, which is expected to play an important role in determining receptor-binding specificity.

Example 4

Expression of Recombinant Lkn-2

Example 4-1

Expression and Purification of Recombinant Lkn-2 in Escherichia Coli

In order to express only mature Lkn-2 as a recombinant protein except a portion estimated to be a signal peptide, the cloned Lkn-2 cDNA in Example 2 was used as a template, and the following PCR primers and Pfu polymerase (Stratagene, USA) were used. PCR amplification of the open reading frame of mature Lkn-2 using:

Forward primer 5'-CGAATTCCATATGCAGTTCACAAATGATGCAGAG-3 ';

Reverse primer 5'-CGCCGCTCGAGTTGAGTAGGGCTTCAGC-3 '.

The amplified DNA fragment was digested with NdeI / XhoI restriction enzyme, cloned into plasmid pET21a (Novagen, USA), and the recombinant plasmid was named pET21a-Lkn-2 and introduced into E. coli XL-1 Blue. The recombinant plasmid pET21a-Lkn-2 prepared as described above expresses recombinant Lkn-2 having one methionine residue at the N-terminus of mature Lkn-2 and six histidines added at the C-terminus.

The transformant thus prepared was named 'Escherichia coli (XL-1 Blue) hMRP-3' and was deposited on November 5, 1996 under the accession number ATCC 98245 to the American Type Culture Collection (ATCC).

After culturing the transformants to express Lkn-2, the inclusion body was obtained and dissolved in 20 ml denatured buffer (6M guanidine-HCl, 20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 4 mM n-octylglucopyranoside) and centrifuged. The separated supernatant was subjected to activated Ni-column (Novagen, USA) chromatography and heparin agarose column (Pharmacia Fine Chemicals, USA) chromatography to obtain Histidine-tagged recombinant Lkn-2. Isolate and purify.

Example 4-2

Expression and Purification of Recombinant Lkn-2 in Insect Cells

In order to express a complete Lkn-2 including a signal peptide as a recombinant protein, Lkn-2 having a PstI restriction enzyme recognition site at the N-terminus of the Lkn-2 cDNA encoding it and an XbaI restriction enzyme recognition site with the C-terminus inserted therein cDNA was PCR amplified. The amplified PCR product was then isolated and inserted into vector PVL 1392 (Invitrogen, USA) digested with PstI / XbaI restriction enzymes to prepare recombinant plasmid PVL 1392-Lkn-2. The Lkn-2 cDNA inserted into the recombinant recombinant plasmid was then transferred to AcNPV by transfecting both the recombinant plasmid and AcNPV (Autographa california nuclear polyhedrosis baculovirus) into Sf-21 insect cells.

AcNPV-Lkn-2 virus plaques are isolated on the basis of their ability to form virion-captured intracellular crystalline proteins (occlusion-negative) and in serum-free Ex-Cell 400 medium (JRH Biosciences, USA). Sf-21 insect cells were grown as infected and used as stock.

Recombinant Lkn-2 was expressed in High five cell line (Invitrogen, USA) cultured in Ex-Cell 400 medium, and expressed Lkn-2 was expressed in HiTrap-Heparin column (Pharmacia Biotech., USA) and HiTrap-SP column (Pharmacia). Biotech., USA).

Example 5

Inhibition of Colony Formation of Bone Marrow Cells by Recombinant Lkn-2

Since some β-chemokines are known to have myelosuppressive effects in laboratory conditions, the colony formation by myeloid progenitor cells present in human bone marrow is purified in Example 4-1. The purpose of this study was to investigate the effect of a recombinant Lkn-2.

That is, low density obtained by centrifugation under Ficoll-Hypaque gradient (1.070gm / cm 3, Sigma Chemical Co., USA) to form colonies of bone marrow cells by colony forming unit-granulocyte-macrophage (CFU-GM) Smear human bone marrow cells at 5 x 10 ⁴ cells / ml in 0.3% agar culture medium containing 10% FBS, and recombinant human granulocyte-macrophage-colony stimulating factor, 100 U / ml, Immunex Corporation, USA ) + rhSLF (recombinant human steel factor, 50ng / ml, Immunex Corporation, USA). On the other hand, in order to form colonies of bone marrow cells by CFU-GM, burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM), electric bone marrow cells are formed. Smear 5 x 10 ⁴ cells / ml in 1% methylcellulose culture medium containing 30% FBS, rhEPO (recombinant human erythropoietin, 1U / ml, Amgen Corporation, USA), rhIL-3 (recombinant human interleukin-3, 100 U / ml, Immunex Corporation, USA) or rhSLF.

After the above stimulus was applied, the cells were cultured in a BNP-210 incubator (Tabai ESPEC Corp., USA) in an atmosphere of 5% CO ₂ and 5% O ₂ , and after 14 days, colony counts were calculated (cf. 4 (A)). , 4 (B) and 4 (C) degrees). At this time, recombinant Lkn-2 was added at 50ng / ml per plate.

FIG. 4 (A) shows the extent to which recombinant Lkn-2 inhibits colonies formed by CFU-GM, and FIG. 4 (B) shows the extent to which recombinant Lkn-2 inhibits colonies formed by CFU-GM. 4 (C) shows the extent to which recombinant Lkn-2 inhibits colonies formed by CFU-GEMM, respectively. In FIGS. 4 (A) -4 (C), the dilution control shows 20 mM Tris-HCl, pH 7.9 with 150 mM NaCl; The control on the X-axis did not contain any serum before or after immunization. As a result, serum before immunization with Lkn-2 was added, and anti-Lkn with anti-serum after immunization with Lkn-2. Each case is shown.

As shown in Figs. 4 (A) to 4 (C), it was found that recombinant Lkn-2 significantly inhibited colony formation, comparable to hMIP-1α, a known colony-inhibiting chemokine; In addition, antiserum specific for Lkn-2 neutralizes the inhibitory effect of colony formation of Lkn-2, while it does not neutralize the inhibitory effect of hMIP-1α colony formation; The pre-immune serum did not have any effect on the inhibitory effect of Lkn-2 or hMIP-1α on colony formation.

Example 6

Investigation of the function of recombinant Lkn-2 as a coin factor

In order to examine the chemotaxis of recombinant Lkn-2, peripheral blood mononuclear cells (PBMC) of healthy humans were first obtained by centrifugation under a Ficoll-Hypaque gradient (1,077 gm / cm 3). Then, monocytes isolation from PBMC was performed as an ability to adhere to the plastic surface, and the cells remaining after such two monocyte separations were obtained as lymphocytes. Purity of the monocytes and lymphocytes obtained as described above was measured by microscopic observation of cytospin stained with Diff-Quick (Baxter Scientific, USA), resulting in 90% and 88%, respectively.

In addition, human neutrophils are obtained by precipitating erythrocytes with 3% Dextran T 500 (Pharmacia Fine Chemicals, UAS), and then centrifuging them under a Ficoll-Hypaque gradient, and obtaining the erythrocytes from a hypotonic solution. Prepared by dissolving at. The purity of the neutrophils thus obtained was determined by morphology and was found to be greater than 95%.

The monocytes and lymphocytes isolated from the above were 2 × 10 ⁶ cells / ml and 8 × 10 ⁶ cells / ml in 0.5% low endotoxin BSA (Sigma Chemical Co., USA) and RPMI 1640 (Gibco, USA) added 20 mM Hepes, respectively. Suspensions in ml and neutrophils were suspended in HBSS at a concentration of 1 x 10 ⁶ cells / ml.

Then, the degree of cell migration in the 48-well microchamber (Neuroprobe, USA) was measured as follows. The lower wells of the microchambers contain buffer only (control) or buffers containing recombinant Lkn-2, hMIP-1α (PeproTech., USA), RANTES (PeproTech., USA) or IL-8 (PeproTech., USA). The solution is filled and the upper wells are filled with 50 μl of the cell suspension described above, and these two layers are fractionated with an appropriate pore filter (without polyvinylpyrrolidone). The pore diameter of the filter used when the cells to be measured were neutrophils and lymphocytes was 3 μm, and the filter diameter used when the monocytes were 5 μm. At 37 ° C. for 1 hour (for neutrophils), 2 hours (for monocytes), 4 hours (for lymphocytes), the microchamber is left, then the filter is removed from the chamber and washed, followed by cell immobilization and Diff-Quick. The cell number was calculated by staining with (Fig. 5 (A) to 5 (C)).

In Figs. 5 (A) to 5 (C), the number obtained by dividing the number of cells transferred to chemokine, which is the test group, by the number of cells moved to the control, was used as a chemoactic index.

5 (A) is a graph showing the chemotaxis of recombinant Lkn-2 and RANTES for lymphocytes, 5 (B) is a graph showing the chemotaxis of recombinant Lkn-2 and hMIP-1α for monocytes, (C) is a graph showing the chemotaxis of recombinant Lkn-2 and IL-8 against neutrophils.

As shown in Figs. 5 (A) to 5 (C), recombinant Lkn-2 was found to be a potent chemotactic factor for human peripheral blood neutrophils, monocytes and lymphocytes. In addition, recombinant Lkn-2 showed a chemotaxis similar to that of RANTES for lymphocytes, and a chemotaxis similar to that of IL-8 for neutrophils.

Example 7

Analysis of Calcium Flux by Recombinant Lkn-2

Example 7-1

Calcium Outflow Analysis in Lymphocytes, Monocytes, and Neutrophils

The aim of this study was to determine whether recombinant Lkn-2 binds to receptors, activates lymphocytes, monocytes and neutrophils to induce calcium efflux from them and how they compete with other agonists for the receptors.

Activation of the receptor was measured by [Ca ²⁺ ] change in purified peripheral blood leukocyte subsets (lymphocytes, monocytes, neutrophils) with an MSIII fluorimeter (Photon Technology International, USA). That is, the cells were reacted with 2 μM fura-2AM (Sigma Chemical Co., USA) for 45 minutes at 37 ° C. and washed twice to give a concentration of 1 × 10 ⁷ cells / ml in HBSS (pH 7.4) containing 0.05% BSA. Suspended. 2 ml of this cell suspension was placed in a stirred, water-jacketed cuvette surrounded by water at 37 ° C. and continuously excited at 340 nm and 380 nm. The fluorescence emitted at this time was measured at 510 nm before and after addition of 25 nM agonist (recombinant Lkn-2, hMIP-1α, RANTES or IL-8) (see Figures 6 (A) to 6 (F)). ).

The relative fluorescence in Figs. 6 (A) to 6 (F) is expressed as the relative ratio of the fluorescence excited at 340 nm and 380 nm.

Figure 6 (A) shows the relative fluorescence measured for a series of stimuli by RANTES and recombinant Lkn-2 applied to lymphocytes; Figure 6 (B) Relative fluorescence measured for a series of stimuli by recombinant Lkn-2 and RANTES applied to lymphocytes; Figure 6 (C) is the relative fluorescence measured for a series of stimuli by hMIP-1α and recombinant Lkn-2 applied to monocytes; Figure 6 (D) is the relative fluorescence measured for a series of stimuli by recombinant Lkn-2 and hMIP-1α applied to monocytes; Figure 6 (E) Relative fluorescence measured for a series of stimuli by IL-8 and recombinant Lkn-2 applied to lymphocytes; Figure 6 (F) shows the relative fluorescence measured for a series of stimuli by recombinant Lkn-2 and IL-8 applied to lymphocytes, respectively.

As shown in FIGS. 6 (A) to 6 (F), recombinant Lkn-2 induced calcium outflow in lymphocytes, monocytes and neutrophils.

On the other hand, when G protein-coupled receptors are continuously exposed to a ligand having the same flow path of the receptor signal within a short time, it is known that the signal sensing ability of the electric receptor is desensitized. This phenomenon has also been shown for subsequent stimulation of recombinant Lkn-2 applied to the receptor expressing cells described above.

In addition, as shown in Figs. 6 (A) to 6 (F), in the event of subsequent stimulation by RANTES and hMIP-1α, recombinant Lkn-2 completely desensitized lymphocytes and monocytes (see 6 (B)). 6 (D)), RANTES and hMIP-1α completely desensitize lymphocytes and monocytes in the event of subsequent stimulation by recombinant Lkn-2 (see FIG. 6 (A) C) can also be seen. This indicates that recombinant Lkn-2 shares a receptor with RANTES and hMIP-1α.

Example 7-2

Calcium efflux analysis in cell lines expressing CC chemokine receptor

In Example 7-1, recombinant Lkn-2 was shown to activate the very receptor activated by RANTES and hMIP-1α. To test this, recombinant Lkn-2 expresses a CC or CXC chemokine receptor. The cell line was examined for activation. Examples herein use HOS cell lines expressing recombinant CCR1, CCR2B, CCR3, CCR4, CCR5 and CXCR4 (AIDS Research and Reference Reagent Program of NIH, USA) instead of using a purified leukocyte subset. The experiment was performed in the same manner as in 7-1. hMIP-1α binds to both CCR1 and CCR5 and RANTES is known to bind to both CCR1, CCR3, CCR5.

As a result, calcium outflow was observed only in HOS cells expressing the CCR1 receptor.

7 (A) or relative fluorescence measured for a series of stimuli by IL-8, hMIP-1α and hMIP-1α applied to HOS cells expressing CCR1 receptor; 7 (B) Relative fluorescence measured for a series of stimuli by hMIP-1α and recombinant Lkn-2 applied to HOS cells expressing CCR1 receptor; 7 (C) shows the relative fluorescence measured for a series of stimuli by recombinant Lkn-2 and hMIP-1α applied to HOS cells expressing the CCR1 receptor, respectively.

As shown in FIGS. 7 (A) to 7 (C), hMIP-1α-mediated Ca ²⁺ signal specificity was observed through CCR1, and recombinant Lkn-2-mediated Ca ²⁺ signal was observed in hMIP-1α-mediated Ca ^{2 +} bar can be seen the Sikkim completely desensitized the signal, which indicates that the recombinant hMIP-1α and Lkn-2 that the use of the CCR1 receptor in the cavity.

From the above results, it can be seen that recombinant Lkn-2 is a stronger agonist for CCR1 than hMIP-1α, which is clearly demonstrated in the results of FIG. 8, which investigated the reaction according to the concentration of recombinant Lkn-2 and hMIP-1α. It is becoming.

As described and demonstrated in detail above, the present invention provides a novel C6 β-chemokine Lkn-2 cDNA isolated from human mononuclear cell lines and an amino acid sequence inferred therefrom. The Lkn-2 cDNA open thaw encodes a total of 137 amino acids, including a signal peptide consisting of 21 amino acids, of which the molecular weight of the mature protein consisting of 116 amino acids is estimated to be 12,500 Daltons. Recombinant Lkn-2, purified by expressing the aforementioned Lkn-2 cDNA in recombinant E. coli or insect cells, significantly inhibits colony formation, has chemotaxis that attracts lymphocytes, monocytes and neutrophils, and hMIP-1α and RANTES It has been shown to bind to the receptor of binding CCR1. The recombinant Lkn-2 protein can be used for antibody production, treatment during HIV-1 infection, bone marrow hepatocyte protection during chemotherapy or radiation therapy, leukemia inhibitor, and the like.

Claims (14)

CDNA and its functional equivalent of C6 β-chemokine Lkn-2 (leukotactin-2) in humans with the following nucleotide sequences:

Lkn-2 (leukotactin-2) protein having the following amino acid sequence or functionally equivalent amino acid sequence deduced from the nucleotide sequence of claim 1:

The method of claim 2, Amino acid sequence 1 to 21 signal peptide (signal peptide) characterized in that Lkn-2 (leukotactin-2) protein. An expression vector comprising a gene from which bases 1 to 63 are removed from the cDNA of claim 1 or the cDNA base sequence of claim 1. The method of claim 1, Expression vector is characterized in that pET21a-Lkn-2 Expression vector. The method of claim 1, Expression vector is characterized in that PVL 1392-Lkn-2 Expression vector. Escherichia coli (XL-1 Blue) hMRP-3 transformed with the expression vector of claim 5 (ATCC 98245). The host cell is transformed with an expression vector comprising the gene of claim 1 or the genes 1 to 63 removed from the gene of claim 1, and then the recombinant Lkn-2 (leukotactin-2) is cultured by culturing the transformant. Method for producing a recombinant Lkn-2 comprising the step of obtaining. The method of claim 8, The method for producing a recombinant Lkn-2, characterized in that the host cell is E. coli or insect cells. The host cell is transformed with an expression vector comprising the gene of claim 1 or the genes 1 to 63 removed from the gene of claim 1, followed by culturing the transformant to recombinant Lkn-2 (leukotactin-2). Recombinant Lkn-2 prepared by a process for obtaining the same. The method of claim 10, Characterized by having the ability to inhibit colony formation Recombinant Lkn-2 (leukotactin-2). The method of claim 10, About human peripheral blood neutrophils, monocytes and lymphocytes Characterized by exhibiting strong chemotaxis Recombinant Lkn-2 (leukotactin-2). The method of claim 10, Characterized by binding to the receptor CCR1 Recombinant Lkn-2 (leukotactin-2). The method of claim 13, hMIP-1α (huamn macrophage inflammatory protein-1α) And RANTES (regulated activated normal T cell expressed sequence) characterized in that sharing the receptor CCR1 Recombinant Lkn-2 (leukotactin-2).

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